Interplay between the overlapping ends of tropomyosin and the N terminus of cardiac troponin T affects tropomyosin states on actin

The functional significance of the molecular swivel at the head-to-tail overlapping ends of contiguous tropomyosin (Tm) dimers in striated muscle is unknown. Contractile measurements were made in muscle fibers from transgenic (TG) mouse hearts that expressed a mutant α-Tm (Tm H276N ). We also reconstituted mouse cardiac troponin T (McTnT) N-terminal deletion mutants, McTnT 1–44 Δ and McTnT 45–74 Δ , into muscle fibers from Tm H276N . For controls, we used the wild-type (WT) McTnT because altered effects could be correlated with the mutant forms of McTnT. Tm H276N slowed crossbridge (XB) detachment rate ( g ) by 19%. McTnT 1–44 Δ attenuated Ca 2+ -activated maximal tension against Tm WT (36%) and Tm H276N (38%), but sped g only against Tm H276N by 35%. The rate of tension redevelopment decreased (17%) only in McTnT 1–44 Δ + Tm H276N fibers. McTnT 45–74 Δ attenuated tension (19%) and myofilament Ca 2+ sensitivity (pCa 50 =5.93 vs. 6.00 in the control fibers) against Tm H276N , but not against Tm WT background. Thus, altered XB cycling kinetics decreased the fraction of strongly bound XBs in McTnT 1–44 Δ + Tm H276N fibers, whereas diminished thin-filament cooperativity attenuated tension in McTnT 45–74 Δ + Tm H276N fibers. In summary, our study is the first to show that the interplay between the N terminus of cTnT and the overlapping ends of contiguous Tm effectuates different states of Tm on the actin filament. —Mamidi, R., Michael, J. J., Muthuchamy, M., Chandra, M. Interplay between the overlapping ends of tropomyosin and the N terminus of cardiac troponin T affects tropomyosin states on actin

Deletion of 1–43 amino acids in cardiac myosin essential light chain blunts length dependency of Ca2+ sensitivity and cross-bridge detachment kinetics

The role of cardiac myosin essential light chain (ELC) in the sarcomere length (SL) dependency of myofilament contractility is unknown. Therefore, mechanical and dynamic contractile properties were measured at SL 1.9 and 2.2 μm in cardiac muscle fibers from two groups of transgenic (Tg) mice: 1 ) Tg-wild-type (WT) mice that expressed WT human ventricular ELC and 2 ) Tg-Δ43 mice that expressed a mutant ELC lacking 1–43 amino acids. In agreement with previous studies, Ca 2+ -activated maximal tension decreased significantly in Tg-Δ43 fibers. pCa 50 (−log 10 [Ca 2+ ] free required for half maximal activation) values at SL of 1.9 μm were 5.64 ± 0.02 and 5.70 ± 0.02 in Tg-WT and Tg-Δ43 fibers, respectively. pCa 50 values at SL of 2.2 μm were 5.70 ± 0.01 and 5.71 ± 0.01 in Tg-WT and Tg-Δ43 fibers, respectively. The SL-mediated increase in the pCa 50 value was statistically significant only in Tg-WT fibers ( P < 0.01), indicating that the SL dependency of myofilament Ca 2+ sensitivity was blunted in Tg-Δ43 fibers. The SL dependency of cross-bridge (XB) detachment kinetics was also blunted in Tg-Δ43 fibers because the decrease in XB detachment kinetics was significant ( P < 0.001) only at SL 1.9 μm. Thus the increased XB dwell time at the short SL augments Ca 2+ sensitivity at short SL and thus blunts SL-mediated increase in myofilament Ca 2+ sensitivity. Our data suggest that the NH 2 -terminal extension of cardiac ELC not only augments the amplitude of force generation, but it also may play a role in mediating the SL dependency of XB detachment kinetics and myofilament Ca 2+ sensitivity

Deletion of 1–43 amino acids in cardiac myosin essential light chain blunts length dependency of Ca 2+

The role of cardiac myosin essential light chain (ELC) in the sarcomere length (SL) dependency of myofilament contractility is unknown. Therefore, mechanical and dynamic contractile properties were measured at SL 1.9 and 2.2 μm in cardiac muscle fibers from two groups of transgenic (Tg) mice: 1) Tg-wild-type (WT) mice that expressed WT human ventricular ELC and 2) Tg-Δ43 mice that expressed a mutant ELC lacking 1–43 amino acids. In agreement with previous studies, Ca(2+)-activated maximal tension decreased significantly in Tg-Δ43 fibers. pCa(50) (−log(10) [Ca(2+)](free) required for half maximal activation) values at SL of 1.9 μm were 5.64 ± 0.02 and 5.70 ± 0.02 in Tg-WT and Tg-Δ43 fibers, respectively. pCa(50) values at SL of 2.2 μm were 5.70 ± 0.01 and 5.71 ± 0.01 in Tg-WT and Tg-Δ43 fibers, respectively. The SL-mediated increase in the pCa(50) value was statistically significant only in Tg-WT fibers (P < 0.01), indicating that the SL dependency of myofilament Ca(2+) sensitivity was blunted in Tg-Δ43 fibers. The SL dependency of cross-bridge (XB) detachment kinetics was also blunted in Tg-Δ43 fibers because the decrease in XB detachment kinetics was significant (P < 0.001) only at SL 1.9 μm. Thus the increased XB dwell time at the short SL augments Ca(2+) sensitivity at short SL and thus blunts SL-mediated increase in myofilament Ca(2+) sensitivity. Our data suggest that the NH(2)-terminal extension of cardiac ELC not only augments the amplitude of force generation, but it also may play a role in mediating the SL dependency of XB detachment kinetics and myofilament Ca(2+) sensitivity

Interplay between the overlapping ends of tropomyosin and the N terminus of cardiac troponin T affects tropomyosin states on actin

The functional significance of the molecular swivel at the head-to-tail overlapping ends of contiguous tropomyosin (Tm) dimers in striated muscle is unknown. Contractile measurements were made in muscle fibers from transgenic (TG) mouse hearts that expressed a mutant α-Tm (Tm(H276N)). We also reconstituted mouse cardiac troponin T (McTnT) N-terminal deletion mutants, McTnT(1–44)(Δ) and McTnT(45–74)(Δ), into muscle fibers from Tm(H276N). For controls, we used the wild-type (WT) McTnT because altered effects could be correlated with the mutant forms of McTnT. Tm(H276N) slowed crossbridge (XB) detachment rate (g) by 19%. McTnT(1–44)(Δ) attenuated Ca(2+)-activated maximal tension against Tm(WT) (36%) and Tm(H276N) (38%), but sped g only against Tm(H276N) by 35%. The rate of tension redevelopment decreased (17%) only in McTnT(1–44)(Δ) + Tm(H276N) fibers. McTnT(45–74)(Δ) attenuated tension (19%) and myofilament Ca(2+) sensitivity (pCa(50)=5.93 vs. 6.00 in the control fibers) against Tm(H276N), but not against Tm(WT) background. Thus, altered XB cycling kinetics decreased the fraction of strongly bound XBs in McTnT(1–44)(Δ) + Tm(H276N) fibers, whereas diminished thin-filament cooperativity attenuated tension in McTnT(45–74)(Δ) + Tm(H276N) fibers. In summary, our study is the first to show that the interplay between the N terminus of cTnT and the overlapping ends of contiguous Tm effectuates different states of Tm on the actin filament. —Mamidi, R., Michael, J. J., Muthuchamy, M., Chandra, M. Interplay between the overlapping ends of tropomyosin and the N terminus of cardiac troponin T affects tropomyosin states on actin